ROLE OF OXIDATIVE/NITROSATIVE STRESS IN FOLATE-PREVENTABLE NTDS Despite the recognition of a complex multigenic basis for neural tube defect (NTD) affected births, NTD expression varies with the geographic region, season, parental socio-economic status and is conspicuously discordant in dizygotic (non-identical) twins. This knowledge points to the importance of gene-environment interactions as a basis for NTD development. Notwithstanding an appreciation for the importance of environmental factors, the list of established teratogenic risk factors is surprisingly limited. Another important knowledge gap is the molecular mechanism by which periconceptual folic acid (FA) supplementation protects against NTDs - this is despite a chain of clinical research that extends over a half- century. Research proposed in project #2 will test the overarching hypothesis that embryonic exposure to reactive oxygen and nitrogen species (RONS) is a unifying environmental basis for disparate genetic and disease associated NTDs. We posit that RONS overexposure during neurulation (where RONS may initially come from multiple potential exogenous and endogenous sources), leads to spatiotemporal perturbations in the synthesis and bioactive lifetime of nitric oxide (NO), a critical factor for successful neurulation. Notably, a fundamental role for NO during neurulation is indicated by findings that the prevalence of NTD-affected conceptuses increased when endogenous NO production by NO synthase (NOS) is inhibited and in mothers who express a nos3 gene polymorphism that has been associated with vascular NO insufficiency. Aim 1 seeks to determine whether biochemical and histochemical measures of enhanced RONS and NO insufficiency are indeed associated with NTD-affected embryos. Aim 2 is to determine whether in murine models, NTDs and RONS can be ameliorated by agents that replete the NOS cofactor tetrahydrobiopterin (BH4) and thereby oppose NOS uncoupling. Additionally, Aim 2 will also test the therapeutic utility of NTD protection by a novel class of superoxide-dependent NO donor molecules that we have shown to protect against BH4 oxidation and N0S3 uncoupling in blood vessels. Aim 3 is to use untargeted profiling for broad discovery of metabolites (and biochemical pathways) that are deranged in NTD-affected conceptuses and reversed by FA supplementation Finally, Aim 4 will test the novel hypothesis that a fundamental mechanism by which FA supplementation protects against NTDs is scavenging of the powerful oxidant, peroxynitrite. RELEVANCE (See instructions): This research seeks to translate breakthrough knowledge that has emerged from the vascular biology field in attempt to: (1) develop new insights into the fundamental molecular basis for environmentally-triggered NTDs, (2) understand why folate supplementation affords protection against NTDs, and (3) establish a novel class of drugs that may reduce the prevalence of NTDs. Another novel aspect ofthis research is our use of untargeted metabolite profiling for discovery of yet unrecognized NTD-associated biochemical perturbations.